Keizo Hayashiya

A virus-inactivating protein isolated from the digestive juice of the silkworm, Bombyx mori

Abstract A protein that can precipitate nuclear polyhedrosis virus (NPV) in vitro was isolated from the digestive juice of silkworm larvae ( Bombyx mori ) by the procedures of gel filtration and ion-exchange and hydroxylapatite column chromatography. The SDS-polyacrylamide gel electrophoretic and the ultracentrifugal analyses showed that the purified substance was a homogenous simple protein. The molecular weight of the purified protein was 27,000–28,000 and the sedimentation coefficient was 2.61 S. This protein had an additional activity to inactivate NPV of B. mori in vitro, somewhat analogous to serological neutralization by serum proteins. Electron microscope observations showed that amorphous materials could be found on the surface of envelopes and that the nucleocapsids disappeared.

Isolation of a protein with saccharase activity from the liquid silk of the silkworm, Bombyx mori

Abstract The present report indicates that a protein having saccharase activity can be purified from the contents of the silk glands (liquid silk) of the silkworm, Bombyx mori. The protein was purified by means of chromatography on Sephadex G-75, DEAE-cellulose and gel filtration on Sephadex G-200 column. The protein was shown to be homogenous by polyacrylamide disc electrophoresis. The enzymatic saccharase properties of this protein are the following: K m of 5.71 m m , an optimum pH of 6.5, a heat of activation at 30°C of 7.45 kcal mole−1 and a transition temperature of about 40°C. The activity of the saccharase is strongly inhibited with p- chloromercuribenzoate and activated by cysteine. The amino acid composition of the protein having saccharase activity is rich in glycine, asparatic acid, and glutamic acid. The molecular weight of the saccharase is estimated to be about 70,000. This purified protein contains about 2.4% carbohydrate. Sucrose is not detected in the liquid silk fraction.

Hydrolysis of sucrose by the cocoon of the silkworm, Bombyx mori

Abstract Saccharase activity, which disappears at the beginning of spinning, appears again in the cocoon. It originates from the liquid silk. Crude proteins containing saccharase are released from the cocoon by treatment with papain and NaHSO3 but saccharase is released from cocoons incubated with distilled water. The Km and optimal pH values of the crude saccharase released from the cocoon is 14.3 mM and 6.5, respectively. It is suggested that soluble saccharase in the liquid silk in vivo transforms insoluble saccharase in the cocoon in vitro.

The Application of Polarographic Method for the Quantitative Micro-Determination of Proteins

(1)蛋白質の定量法の一つであるビウレット法の改良法として,蛋白質溶液の1N NaOH溶液に,正確に一定量の硫酸銅を含ませ,錯塩形成後の過剰の遊離の銅量をポーラログラフ法によって定量し,銅と結合した蛋白質量を間接的に測定する方法について研究した. (2)蛋白質濃度が8×10-2mg/ml以下で,硫酸銅濃度が2×10-4M以下という微量定量の場合には,蛋白質と銅とは定量的に結合し,かつ,ポーラログラフ法による過剰の銅量の測定に,何ら支障がみられない. (3) a)蛋白質銅錯塩の還元波は,銅の還元波の電位より負にずれるために両還元波を分離して測定でき, b)いわゆる高分子物質などによる銅の波高の低下も,この濃度では無視され, c)アミノ酸,糖類などの不純物が存在する場合にも,その影響をうけない. d)蛋白質濃度が1×10-2～8×10-2mg/mlの範囲が最も適当な測定範囲で微量定量が可能であり, e)蛋白質の種類による定量値の差異もほとんどみられない.